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Identification of the PLK2-dependent phosphopeptidome by quantitative proteomics [corrected].

Franchin C, Cesaro L, Pinna LA, Arrigoni G, Salvi M - PLoS ONE (2014)

Bottom Line: Stable isotope labeling based quantitative phosphoproteomics was applied to identify the phosphosites generated by PLK2.A total of 98 unique PLK2-dependent phosphosites from 89 proteins were identified by LC-MS/MS.Analysis of the primary structure of the identified phosphosites allowed the detailed definition of the kinase specificity and the compilation of a list of potential PLK2 targets among those retrieved in PhosphositePlus, a curated database of in cell/vivo phosphorylation sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, University of Padova, Padova, Italy; Proteomics Center of Padova University, Padova, Italy.

ABSTRACT
Polo-like kinase 2 (PLK2) has been recently recognized as the major enzyme responsible for phosphorylation of α-synuclein at S129 in vitro and in vivo, suggesting that this kinase may play a key role in the pathogenesis of Parkinson's disease and other synucleinopathies. Moreover PLK2 seems to be implicated in cell division, oncogenesis, and synaptic regulation of the brain. However little is known about the phosphoproteome generated by PLK2 and, consequently the overall impact of PLK2 on cellular signaling. To fill this gap we exploited an approach based on in vitro kinase assay and quantitative phosphoproteomics. A proteome-derived peptide library obtained by digestion of undifferentiated human neuroblastoma cell line was exhaustively dephosphorylated by lambda phosphatase followed by incubation with or without PLK2 recombinant kinase. Stable isotope labeling based quantitative phosphoproteomics was applied to identify the phosphosites generated by PLK2. A total of 98 unique PLK2-dependent phosphosites from 89 proteins were identified by LC-MS/MS. Analysis of the primary structure of the identified phosphosites allowed the detailed definition of the kinase specificity and the compilation of a list of potential PLK2 targets among those retrieved in PhosphositePlus, a curated database of in cell/vivo phosphorylation sites.

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Putative PLK2-substrate localization (A) and functional (B) analysis.Subcellular localization (A) and functional analysis (B) for each protein have been assigned using GeneCoDis3 webserver [35], [36].
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pone-0111018-g006: Putative PLK2-substrate localization (A) and functional (B) analysis.Subcellular localization (A) and functional analysis (B) for each protein have been assigned using GeneCoDis3 webserver [35], [36].

Mentions: Figure 6 shows the analysis of subcellular localization (A) and molecular functions (B) of putative PLK2 substrates identified in this study. Identified proteins localize both in cytoplasmic and nuclear compartments and participate to several processes where the involvement of PLK2 kinase has not been described yet. As mentioned above the number of bona fide PLK2 substrates identified so far is low and includes not only cytosolic proteins, but also plasma membrane [32] and nuclear [33] substrates. The localization of PLK2 at centrosomes where it regulates centriole duplication, has been deeply investigated [4]. However PLK2 has been identified also in different subcellular compartments, such as cytoplasm, nucleus (PLK2 contains a nuclear localization signal [34]), and membranes in HEK 293T cells [12], while in primary hippocampal neurons PLK2 shows primarily a nuclear localization [12]. Co-localization between the kinase and its putative substrates suggests unanticipated regulatory roles for PLK2 in nuclear functions.


Identification of the PLK2-dependent phosphopeptidome by quantitative proteomics [corrected].

Franchin C, Cesaro L, Pinna LA, Arrigoni G, Salvi M - PLoS ONE (2014)

Putative PLK2-substrate localization (A) and functional (B) analysis.Subcellular localization (A) and functional analysis (B) for each protein have been assigned using GeneCoDis3 webserver [35], [36].
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4206460&req=5

pone-0111018-g006: Putative PLK2-substrate localization (A) and functional (B) analysis.Subcellular localization (A) and functional analysis (B) for each protein have been assigned using GeneCoDis3 webserver [35], [36].
Mentions: Figure 6 shows the analysis of subcellular localization (A) and molecular functions (B) of putative PLK2 substrates identified in this study. Identified proteins localize both in cytoplasmic and nuclear compartments and participate to several processes where the involvement of PLK2 kinase has not been described yet. As mentioned above the number of bona fide PLK2 substrates identified so far is low and includes not only cytosolic proteins, but also plasma membrane [32] and nuclear [33] substrates. The localization of PLK2 at centrosomes where it regulates centriole duplication, has been deeply investigated [4]. However PLK2 has been identified also in different subcellular compartments, such as cytoplasm, nucleus (PLK2 contains a nuclear localization signal [34]), and membranes in HEK 293T cells [12], while in primary hippocampal neurons PLK2 shows primarily a nuclear localization [12]. Co-localization between the kinase and its putative substrates suggests unanticipated regulatory roles for PLK2 in nuclear functions.

Bottom Line: Stable isotope labeling based quantitative phosphoproteomics was applied to identify the phosphosites generated by PLK2.A total of 98 unique PLK2-dependent phosphosites from 89 proteins were identified by LC-MS/MS.Analysis of the primary structure of the identified phosphosites allowed the detailed definition of the kinase specificity and the compilation of a list of potential PLK2 targets among those retrieved in PhosphositePlus, a curated database of in cell/vivo phosphorylation sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, University of Padova, Padova, Italy; Proteomics Center of Padova University, Padova, Italy.

ABSTRACT
Polo-like kinase 2 (PLK2) has been recently recognized as the major enzyme responsible for phosphorylation of α-synuclein at S129 in vitro and in vivo, suggesting that this kinase may play a key role in the pathogenesis of Parkinson's disease and other synucleinopathies. Moreover PLK2 seems to be implicated in cell division, oncogenesis, and synaptic regulation of the brain. However little is known about the phosphoproteome generated by PLK2 and, consequently the overall impact of PLK2 on cellular signaling. To fill this gap we exploited an approach based on in vitro kinase assay and quantitative phosphoproteomics. A proteome-derived peptide library obtained by digestion of undifferentiated human neuroblastoma cell line was exhaustively dephosphorylated by lambda phosphatase followed by incubation with or without PLK2 recombinant kinase. Stable isotope labeling based quantitative phosphoproteomics was applied to identify the phosphosites generated by PLK2. A total of 98 unique PLK2-dependent phosphosites from 89 proteins were identified by LC-MS/MS. Analysis of the primary structure of the identified phosphosites allowed the detailed definition of the kinase specificity and the compilation of a list of potential PLK2 targets among those retrieved in PhosphositePlus, a curated database of in cell/vivo phosphorylation sites.

Show MeSH
Related in: MedlinePlus